Epidermal growth factor, hereinafter referred to as EGF, is a biologically potent, monomeric protein produced naturally by many species of mammals including humans. It stimulates the growth of new skin and other epithelial tissues and thus has great potential as a therapeutic agent in corneal transplant healing, in skin regeneration e.g. for treating burns and grafts, and in wound healing e.g. for treating surgical incisions and stomach and other ulcers.
A genetic engineering approach has been adopted to produce EGF in amounts sufficient to meet commercial demands, although difficulties have been encountered. Like many other proteins produced nakedly in recombinant microbial hosts, EGF is degraded intracellularly by the action of native proteases and only modest yields are attainable. To protect EGF from protease action and elevate yields, EGF is now typically produced as a fusion protein, bound to a stabilizing carrier protein that inhibits EGF degradation (see, for example, EP 234,888). For use in therapeutic applications, the EGF is liberated from the carrier protein and then purified. In economic terms, the advantage of increased yields offered by the fusion protein technology must be weighed against the disadvantage of the laborious downstream processing required to release and recover EGF in its authentic, monomeric form.
An alternative strategy for producing protease-sensitive proteins in microbial hosts has been proposed in the literature. According to this proposal, the host is transformed to express the desired protein in a multimeric form that comprises repeating units of the desired protein linked tandemly through a cleavage-sensitive site. Following recovery of the protein multimer, the desired monomeric form of the protein is liberated for pharmaceutical use by digestion with the appropriate cleavage agent, usually a site-specific enzyme. The general concept is described in Canadian patent 1,213,537. It has been suggested that EGF may be produced using this strategy (see GB 2,172,890).
The strategy of producing EGF either as a fusion protein or as a multimer is attractive in the sense that the EGF is rendered more stable in the microbial host. However, because its biological activity can be eliminated or significantly impaired when it is bound to other protein moieties, time consuming efforts are employed to recover and purify EGF per se from the hybrid protein. Moreover, it is often necessary to modify the structure of EGF so that the selected cleavage site remains unique, and the EGF molecule is not cleaved internally by the technique designed to liberate it.
It is an object of the present invention to provide a method for producing proteins having the biological activity of EGF.
It is another object of the present invention to provide biologically active variants of EGF.
It is a further object of the present invention to provide pharmaceutical compositions useful to promote wound healing.
It is another object of the present invention to provide a method for treating wounds to promote healing thereof.